1. Field of the Invention
The present invention relates to a polypeptide as a novel fusion protein, a polynucleotide encoding the polypeptide, a vector comprising the polynucleotide, a transformed cell comprising the vector, and a therapeutic agent for cancer.
2. Background Art
Several cancer-related genes have been known so far. In particular, tyrosine kinase genes, which encode important enzymes directly regulating cell growth, have been known to be activated even by substitution or deletion in amino acid sequences and thereby bring about carcinogenesis.
For example, NPM-ALK fusion genes encoding NPM fused with ALK tyrosine kinase are observed in more than half of the cases of anaplastic large-cell lymphoma (ALCL) and the activation of ALK kinase has been shown to be important for tumor cell growth by NPM-ALK (“Science”, (US), 1997, Vol. 278, p. 1309-1312, and “Science”, (US), 1994, Vol. 263, p. 1281-1284).
The presence of a new type of abnormal kinase has been reported to be found in approximately 10% of lung cancer cases, and this report, however, has made no reference to specific molecules (Proceedings of the 65th Annual Meeting of the Japanese Cancer Association, O-324 (issued on Aug. 28, 2006)).
The echinoderm microtubule-associated protein like protein (EML4) (GenBank accession Number: NM—019063) has a basic region at the amino terminus, and further has carboxyl-terminal WD domains (“Genomics”, (US), 2000, Vol. 68, p. 348-350). The physiological functions of EML4 have been little known.
On the other hand, Anaplastic Lymphoma Kinase (ALK) (GenBank accession Number: AB209477) is receptor tyrosine kinase (Oncogene. Jan. 30, 1997; 14 (4): 439-49). The ALK has a transmembrane domain in the middle, the carboxyl-terminal tyrosine kinase domain and amino terminal outer membrane domain(Oncogene. Jan. 30, 1997; 14 (4): 439-49).
Full-length ALK expression has been reported so far in some cancer cells of ectodermal origin, such as neuroblastoma, glioblastoma, breast cancer, and melanoma (the full-length ALK expression has not been observed in cancer cells of endodermal and mesodermal origins) (“International journal of cancer”, (US), 2002, Vol. 100, p. 49-56). Full-length ALK is expressed in many neuroblastoma cell lines. However, the autophosphorylation of ALK is not observed in these neuroblastoma cell lines. Moreover, ALK expression has been reported, from the cohort analysis of neuroblastoma patients, to be weakly associated with cancer. It has been suggested that ALK expression in neuroblastoma may reflect its expression in normal neural differentiation, rather than its association with cancer (“Cellular and molecular life sciences”, (Switzerland), 2004, Vol. 61, p. 2939-2953). On the other hand, in reported cases, ligands such as pleiotrophin and midkine as well as the gene amplification of ALK itself increase the autophosphorylation of ALK and mobilize intracellular signals. It has also been reported that ALK may contribute to cancer cell growth (“Journal of cellular physiology”, (US), 2004, Vol. 199, p. 330-358).
In some cases of human malignant lymphoma and inflammatory myofibroblastic tumor, the ALK gene has been reported to be fused with other genes (NPM, CLTCL, TFG, CARS, SEC31L1, etc.) as a result of chromosomal translocation or inversion and thereby form a fusion type of tyrosine kinase (“Oncogene 9”: 1567-1574, 1994, “Am J Pathol” 160: 1487-1494, 2002, “Science”, (US), 1994, Vol. 263, p. 1281-1284, “Blood”, (US), 1995, Vol. 86, p. 1954-1960, “Blood”, (US), 2000, Vol. 95, p. 3204-3207, “Blood”, (US), 1999, Vol. 94, p. 3265-3268, “Laboratory investigation; a journal of technical methods and pathology”, (US), 2003, Vol. 83, p. 1255-1265, “International journal of cancer”, (US), 2006, Vol. 118, p. 1181-1186, “Am J Pathol” 157: 377-384, 2000, “Blood” 90: 2901-2910, 1997, “Am J Pathol”. 2000 Mar; 156 (3): 781-9). Moreover, a method for identifying a protein as a fusion partner for ALK using ALK antibodies has been reported (“PNAS” 2006 103, 7402-7407). On the other hand, a fusion gene of EML4 and ALK has not been reported. Since most partner molecules have a complex formation domain, the fusion protein itself has been thought to form a complex. This complex formation has been considered to cause loss of control of the tyrosine kinase activity of ALK and induce carcinogenesis with abnormally activated intracellular signals (“Cellular and molecular life sciences”, (Switzerland), 2004, Vol. 61, p. 2939-2953). Indeed, it has been reported that the use of ALK shRNA or ALK kinase-inhibiting compound for lymphoma cells expressing ALK fusion proteins can induce cell growth inhibition and cell death. ALK inhibitor have been reported to inhibit lymphoma (PNAS, Jan, 2, 2007, 104 (1), 270-275 Epub2006 Dec). Therefore, it has been suggested that the ALK fusion protein may serve as a therapeutic target for lymphoma and inflammatory myofibroblastic tumor (“Blood”, (US), 2006, Vol. 107, p. 689-697, “Blood”, (US), 2006, Vol. 107, p. 1617-1623, “Laboratory investigation; a journal of technical methods and pathology”, (US), 2005, Vol. 85, p. 1544-1554). It has also been suggested that ALK may serve as a therapeutic target for other cancers whose growth involves ALK as described above (“Blood”, (US), 2006, Vol. 107, p. 1617-1623, “Laboratory investigation; a journal of technical methods and pathology”, (US), 2005, Vol. 85, p. 1544-1554).
It has been reported that WHI-P131 and WHI-P154, which have been utilized as JAK3 tyrosine kinase-inhibiting substances, inhibit the activity of NPM-ALK (“Laboratory investigation; a journal of technical methods and pathology”, (US), 2005, Vol. 85, p. 1544-1554). It has also been reported that low-molecular-weight ALK inhibitor induces the cell death of NPM-ALK-expressing lymphoma cell lines (“Blood”, (US), 2006, Vol. 107, p. 1617-1623). In addition, plural low-molecular-weight compounds having an inhibitory activity against ALK have been reported so far (“Journal of medicinal chemistry”, (US), 2006, Vol. 49, p. 1006-1015, “J Comb Chem.” 8: 401-409, 2006, WO 2004/080980, WO 2005/009389, WO 2005/016894).